1. Field of the Invention
The present invention relates generally to the fields of stem cells and differentiated cells. More particularly, it concerns the generation of induced keratinocyte stem cells (iKCs) from undifferentiated cells and methods of culturing primary keratinocytes.
2. Description of Related Art
Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSC) are capable of long-term proliferation in vitro, while retaining the potential to differentiate into all cell types of the body, including keratinocytes. Thus, these pluripotent cells have the potential to provide an unlimited supply of patient-specific functional keratinocytes for both transplantation therapies and cosmetics/drug development.
To date, several groups have reported procedures to differentiate mouse and human ES/iPS cells to epidermal keratinocytes (cytokeratin 14-positive). Metallo et al. (2010) used retinoic acid and BMP4 on embryoid bodies and mono-layer culture on collagen IV-coated surface without feeder cells to induce keratinocyte differentiation from human ES cells. Kawasaki et al. (2000) reported a method using feeder cells promote neural differentiation of mouse ES cells, and that BMP4 addition promotes the initiation of epidermal determination from neuronal ectoderm. Lian et al. (2013) described a method using small molecule inhibitor of Src family kinases to derive simple epithelial progenitors, which further differentiate to epidermal keratinocytes in serum-free conditions. However, none of these methods have generated epidermal keratinocyte stem cells (cytokeratin 14 and cytokeratin 15 double positive) with a proliferative capacity of more than two population doublings or long-term engraftability.